Many flaviviruses and alphaviruses are serious human pathogens. Some are listed by the NIH and CDC as potential bio-weapons. Understanding the functions of virally encoded proteins is important to developing treatments or preventatives against infection. In addition to their structural proteins, these single-stranded RNA viruses encode enzymes and other proteins that are essential to virus survival, including an RNA-dependent RNA polymerase, a protease with specificity for viral polyproteins, a helicase, and enzymes catalyzing steps in the RNA 5'-capping process. These and other nonstructural proteins work together to process viral polyproteins, to synthesize minus- and plus-strand RNAs in a coordinated manner, to cap viral RNAs, and to deliver viral RNAs to the capsid proteins for packaging. At present, three-dimensional structures are available for only two domains of flavivirus nonstructural proteins; no alphavirus nonstructural protein structures have been reported. The goal of this Project is to determine crystal structures for all soluble, nonstructural proteins of the eleven flaviviruses and nine alphaviruses being studied in the Program Project. The proteins include flavivirus NS1, NS3, and NS5, and alphavirus nsP1, nsP2, nsP3, and nsP4. The protein targets will be studied in parallel using high-throughput technology in order to find optimal constructs and conditions for expression, protein purification, and protein crystallization. Preliminary studies have shown that most of the target proteins can be produced in bacterial expression systems; baculovirus-infected cells will be used for proteins that cannot be produced in bacteria. A second goal of the Project is to understand the structural basis for differences in the behaviors of individual flaviviruses and alphaviruses by comparing structures of each protein from several viruses. This is essential to development of virus-specific inhibitors. Finally, the Project will address specific biological questions about the function of nonstructural proteins, particularly how they are influenced by interactions with other proteins. The Project is designed to accelerate progress towards a full understanding of the virus life cycle at the molecular level. It will also provide invaluable reagents for other Projects in the Program Project.